Nutritional Composition To Promote Healthy Development And Growth

ABSTRACT

A nutritional formula which includes a lipid or fat; a protein source; about 5 to about 100 mg/100 kcal of a source of long chain polyunsaturated fatty acids which comprises docosahexanoic acid; about 0.1 to about 1 mg/100 kcal of a prebiotic composition, wherein the prebiotic composition comprises at least 20% of an oligosaccharide which comprises galacto-oligosaccharide; and about 0.015 to about 0.1 (pg/μg) ppm of TGF-β.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 from (a) copendingand commonly assigned U.S. patent application Ser. No. 12/371,100,entitled Nutritional Composition With Improved Digestibility, filed Feb.13, 2009, which in turn claims priority under 35 U.S.C. §119(e) fromU.S. Provisional Patent Application Ser. No. 61/108,303 filed Oct. 24,2008 and 61/111,009 filed Nov. 4, 2008, and (b) copending and commonlyassigned U.S. patent application Ser. No. 11/172,123, entitled MethodFor Simulating The Functional Attributes of Human Milk OligosaccharidesIn Formula-Fed Infants, filed Jun. 30, 2005, the disclosures of each ofwhich are incorporated by reference herein.

TECHNICAL FIELD

This disclosure relates generally to the field of nutritionalcompositions. More particularly, the disclosure relates to a nutritionalcomposition which provides improved digestibility, as compared toconventional compositions.

BACKGROUND

The gut microflora of a human is a complex collection of interrelatedmicrobes which act together to facilitate the digestive process. In thecase of infants, the gut microflora is rapidly established in the firstfew weeks following birth. The nature of this intestinal colonization isinitially determined by early exposure to environmental sources ofmicrobes as well as the health of the infant. Whether the infant isbreast-fed or formula fed also has a strong influence on the intestinalbacterial population.

In the breast-fed infant, for example, Bifidobacterium spp. dominateamong intestinal bacteria, with Streptococcus spp. and Lactobacillusspp. as less common contributors. In contrast, the microflora offormula-fed infants is more diverse, containing Bifidobacterium spp. andBacteroides spp. as well as the more pathogenic species, Staphylococcus,Escherichia coli and Clostridia. The varied species of Bifidobacteriumin the stools of breast-fed and formula-fed infants differ as well.

Bifidobacteria are generally considered “beneficial” bacteria and areknown to protect against colonization by pathogenic bacteria. Thislikely occurs through competition for cell surface receptors,competition for essential nutrients, production of anti-microbialagents, and production of inhibitory compounds such as short chain fattyacids (SCFA) which may decrease fecal pH and inhibit potentiallypathogenic bacteria.

Bifidobacteria are also associated with resistance to gastrointestinal(GI) tract and respiratory infection as well as an enhanced immunefunction, especially in children and infants. Therefore, the promotionof an intestinal environment in which Bifidobacteria dominate has becomea goal in the development of nutritional compositions, includingnutritional formulations for adults and children and compositions forformula-fed infants.

Human milk (HM) contains a number of factors that may contribute to thegrowth and population of Bifidobacteria in the gut microflora ofinfants. Among these factors is a complex mixture of more than 130different oligosaccharides that reach levels as high as 8-12 g/L intransitional and mature milk. Kunz, et al., Oligosaccharides in HumanMilk: Structure, Functional, and Metabolic Aspects, Ann. Rev. Nutr. 20:699-722 (2000). These oligosaccharides are resistant to enzymaticdigestion in the upper gastrointestinal tract and reach the colonintact, where they serve as substrates for colonic fermentation.

HM oligosaccharides are believed to elicit an increase in the number ofBifidobacteria in the colonic flora, along with a reduction in thenumber of potentially pathogenic bacteria. Kunz, et al.,Oligosaccharides in Human Milk: Structure, Functional, and MetabolicAspects, Ann. Rev. Nutr. 20: 699-722 (2000); Newburg, Do the BindingProperties of Oligosaccharides in Milk Protect Human Infants fromGastrointestinal Bacteria?, J. Nutr. 217:S980-S984 (1997). One way thatHM oligosaccharides may increase the number of Bifidobacteria and reducethe number of potentially pathogenic bacteria is by acting ascompetitive receptors and inhibiting the binding of pathogens to thecell surface. Rivero-Urgell, et al., Oligosaccharides: Application inInfant Food, Early Hum. Dev. 65(S):43-52 (2001).

In addition to reducing the number of pathogenic bacteria and promotingthe population of Bifidobacteria, when HM oligosaccharides arefermented, they produce SCFAs such as acetic, propionic and butyricacids. These SCFAs are believed to contribute to caloric content, serveas a major energy source for the intestinal epithelium, stimulate sodiumand water absorption in the colon, and enhance small bowel digestion andabsorption. In addition, SCFA are believed to contribute to overallgastrointestinal health by modulating gastrointestinal development andimmune function.

The fermentation of HM oligosaccharides also reduces fecal ammonia,amine, and phenol concentrations, which have been implicated as themajor odorous components of feces. Cummings & Macfarlane, The Controland Consequences of Bacterial Fermentation in the Human Colon, J. Appl.Bacteriol. 70:443-459 (1991); Miner & Hazen, Ammonia and Amines:Components of Swine-Building Odor ASAE 12:772-774 (1969); Spoelstra,Origin of Objectionable Components in Piggery Wastes and the Possibilityof Applying Indicator Components for Studying Odour Development, Agric.Environ. 5:241-260 (1980); O'Neill & Phillips, A Review of the Controlof Odor Nuisance from Livestock Buildings: Part 3. Properties of theOdorous Substances which have been Identified in Livestock Wastes or inthe Air Around them J. Agric. Eng. Res. 53:23-50 (1992).

As a result of the oligosaccharides present in HM, the SCFA profile of abreast-fed infant is very different from that of a formula-fed infant.For example, breast-fed infants produce virtually no butyrate, withacetate comprising approximately 96% of the total SCFA production.Lifschitz, et al., Characterization of Carbohydrate Fermentation inFeces of Formula-Fed and Breast-Fed Infants, Pediatr. Res. 27:165-169(1990); Siigur, et al., Faecal Short-Chain Fatty Acids in Breast-Fed andBottle-Fed Infants. Acta. Paediatr. 82:536-538 (1993); Edwards, et al.,Faecal Short-Chain Fatty Acids in Breast-Fed and Formula-Fed Babies,Acta. Paediatr. 72:459-462 (1994); Parrett & Edwards, In VitroFermentation of Carbohydrates by Breast Fed and Formula Fed Infants,Arch. Dis. Child 76:249-253 (1997). In contrast, while formula-fedinfants also have acetate (74%) as the major SCFA in feces, they haveconsiderable amounts of propionate (23%) and small amounts of butyrate(3%) present as well. These differences between the SCFA profiles ofbreast-fed infants and formula-fed infants could affect the energy,digestion, and overall health of the formula-fed infant.

Because cow's milk and commercially available infant formulas that arebased on cow's milk provide only trace amounts of oligosaccharides,prebiotics are often used to supplement the diet of formula-fed infants.Prebiotics have been defined as “non-digestible food ingredients thatbeneficially affect the host by selectively stimulating the growthand/or activity of one or a limited number of bacteria in the colon thatcan improve the health of the host”. Gibson, G. R. & Roberfroid, M. B.,Dietary Modulation of the Human Colonic Microbiota-Introducing theConcept of Probiotics, J. Nutr. 125:1401-1412 (1995). Common prebioticsinclude fructo-oligosaccharide, gluco-oligosaccharide,galacto-oligosaccharide, isomalto-oligosaccharide, xylo-oligosaccharideand lactulose.

The incorporation of various prebiotic ingredients into infant formulashas been disclosed. For example, U.S. Patent App. No. 2003/0072865 toBindels, et al. discloses an infant formula with an improved proteincontent and at least one prebiotic. The prebiotic component can belacto-N-tetaose, lacto-N-fuco-pentaose, lactulose (LOS), lactosucrose,raffinose, galacto-oligosaccharide (GOS), fructo-oligosaccharide (FOS),oligosaccharides derived from soybean polysaccharides, mannose-basedoligosaccharides, arabino-oligosaccharides, xylo-oligosaccharides,isomalto-oligo-saccharides, glucans, sialyl oligosaccharides, andfuco-oligosaccharides.

Similarly, U.S. Patent App. No. 2004/0191234 to Haschke discloses amethod for enhancing the immune response which comprises administeringat least one prebiotic. The prebiotic can be an oligosaccharide producedfrom glucose, galactose, xylose, maltose, sucrose, lactose, starch,xylan, hemicellulose, inulin, or a mixture thereof. The prebiotic can bepresent in an infant cereal.

In addition, other factors present in human breast milk are believed tobe beneficial to the developing body. For instance, functional proteinssuch as transforming growth factor-beta (TGF-β) play a significant rolein many processes necessary for health and development, in infants andchildren, as well as adults.

More specifically, TGF-β is the general name for a family ofpolypeptides, the members of which have multifunctional regulatoryactivities. Three differentially regulated mammalian isoforms (termedTGF-β1, TGF-β2 and TGF-β3) play important roles in a multitude ofprocesses in the developing infant, child and adult. TGF-β is a 25-kDahomodimeric cytokine known to mediate pleitropic functions both withinthe immune system and systemically, it is expressed in several celltypes in the intestinal mucosal including lymphocytes, epithelial cells,macrophages, and stromal cells as well as by T-cells, neutrophils,macrophages, epithelial cells, fibroblasts, platelets, osteoblasts,osteoclasts and others. In addition, TGF-β is present in human breastmilk and may influence multiple aspects of infant health anddevelopment.

TGF-βs are synthesized as large precursor proteins which consist of anamino-terminal pro-domain, comprising a signal sequence andlatency-associated complex, and a mature carboxy-terminal subunit.Biologically active TGF-β are homodimers which consist of two identical,disulfide-linked mature subunits. Release of the TGF-β homodimer fromthe latency-associated complex is necessary for TGF-β to exertbiological activity on target cells. The nature of thelatency-associated complex and the mechanisms responsible for TGF-βrelease are key to understanding TGF-β biological activity in vivo. Inthe human gut, this may be accomplished by the action of proteolyticenzymes, pH extremes, heat, calcium, and/or mechanical tearing.

Accordingly, it would be beneficial to provide a nutritional compositionwhich provides a combination of nutrients designed to encourage healthydevelopment and growth, especially in an infant. Included in thenutritional composition should be a prebiotic substance that simulatesthe functional attributes of human milk oligosaccharides in infants,such as an increase in the population and species of beneficial bacteriain the infant gut and production of a SCFA profile similar to that of abreast-fed infant, and materials which provide a dietary source ofbioactive TGF-β. Additionally, the nutritional composition should bewell tolerated in animals, especially human infants and should notproduce or cause excess gas, abdominal distension, bloating or diarrhea.

BRIEF SUMMARY

Briefly, the present disclosure is directed, in an embodiment, to anutritional composition comprising a lipid or fat, a protein source, asource of long chain polyunsaturated fatty acids which includedocosahexanoic acid (DHA), a prebiotic composition having at least 20%of an oligosaccharide which comprises galacto-oligosaccharide, andTGF-β. In certain embodiments, the prebiotic comprises a combination ofgalacto-oligosaccharide and polydextrose.

The invention is also directed to a nutritional composition comprising:

a. up to about 7 g/100 kcal of a fat or lipid, more preferably about 3to about 7 g/100 kcal of a fat or lipid;

b. up to about 5 g/100 kcal of a protein source, more preferably about 1to about 5 g/100 kcal of a protein source;

c. about 5 to about 100 mg/100 kcal of a source of long chainpolyunsaturated fatty acids which include DHA, more preferably about 10to about 50 mg/100 kcal of a source of long chain polyunsaturated fattyacids which include DHA;

d. about 1.0 to about 10.0 g/L of a prebiotic composition having atleast 20% of an oligosaccharide which comprises galacto-oligosaccharide,more preferably about 2.0 g/L to about 8.0 g/L of a prebioticcomposition having at least 20% of an oligosaccharide which comprisesgalacto-oligosaccharide; and

e. about 0.015 to about 0.1 (pg/μg) ppm TGF-β, more preferably about0.0225 to about 0.075 (pg/βg) ppm TGF-β.

In yet another embodiment, the invention is directed to a nutritionalcomposition having improved digestibility, the composition comprising alipid or fat, a protein source, a source of long chain polyunsaturatedfatty acids which include docosahexanoic acid (DHA), a prebioticcomposition having at least 20% of an oligosaccharide which comprisesgalacto-oligosaccharide, and TGF-β.

Additionally, an embodiment is directed to a nutritional compositionhaving enhanced TGF-β bioactivity, the composition comprising a lipid orfat, a protein source, a source of long chain polyunsaturated fattyacids which include docosahexanoic acid (DHA), a prebiotic compositionhaving at least 20% of an oligosaccharide which comprisesgalacto-oligosaccharide, and TGF-β.

DETAILED DESCRIPTION

The technical problem to be solved by the present invention is toprovide novel nutritional products that are easily digested, providephysiochemical benefits, and/or provide physiological benefits. In anembodiment of the present invention, a nutritional composition comprisesa lipid or fat, a protein source, a source of long chain polyunsaturatedfatty acids which include docosahexanoic acid (DHA), a prebioticcomposition having at least 20% of an oligosaccharide which comprises amixture of D-glucose and D-galactose (commonly referred to asgalacto-oligosaccharide or trans-galacto-oligosaccharide, or GOS), andTGF-β. In certain embodiments, the prebiotic comprises a combination ofgalacto-oligosaccharide and polydextrose. More particularly, thecomposition disclosed herein comprises:

a. up to about 7 g/100 kcal of a fat or lipid, more preferably about 3to about 7 g/100 kcal of a fat or lipid;

b. up to about 5 g/100 kcal of a protein source, more preferably about 1to about 5 g/100 kcal of a protein source;

c. about 5 to about 100 mg/100 kcal of a source of long chainpolyunsaturated fatty acids which include DHA, more preferably about 10to about 50 mg/100 kcal of a source of long chain polyunsaturated fattyacids which include DHA;

d. about 1.0 to about 10.0 g/L of a prebiotic composition having atleast 20% of an oligosaccharide which comprises galacto-oligosaccharide,more preferably about 2.0 g/L to about 8.0 g/L of a prebioticcomposition having at least 20% of galacto-oligosaccharide; and

e. about 0.015 to about 0.1 (pg/μg) ppm TGF-β, more preferably about0.0225 to about 0.075 (pg/μg) ppm TGF-β.

In some embodiments, the nutritional composition may be an infantformula. As used herein, the term “infant” means a person not more than12 months of age. The term “infant formula” applies to a composition inliquid or powdered form that satisfies the nutrient requirements of aninfant by being a substitute for human milk. In the United States, thecontent of an infant formula is dictated by the federal regulations setforth at 21 C.F.R. §§100, 106 and 107. These regulations definemacronutrient, vitamin, mineral, and other ingredient levels in aneffort to simulate the nutritional and other properties of human breastmilk. In a separate embodiment, the nutritional composition may be ahuman milk fortifier, meaning it is a composition which is added tohuman milk in order to enhance the nutritional value of human milk. As ahuman milk fortifier, the inventive composition may be in powder orliquid form. In yet another embodiment, the inventive nutritionalproduct may be a children's nutritional composition. The term “youngchild” or “young children” as used herein means persons more than 12months of age up to the age of three years (36 months). The term “child”or “children” as used herein means persons over the age of 3 years andprior to adolescence.

The nutritional products of the invention may provide minimal, partial,or total nutritional support. The compositions may be nutritionalsupplements or meal replacements. In some embodiments, the compositionsmay be administered in conjunction with a food or nutritionalcomposition. In this embodiment, the compositions can either beintermixed with the food or other nutritional compositions prior toingestion by the subject or can be administered to the subject eitherbefore or after ingestion of a food or nutritional composition. Thecompositions may be administered to preterm infants receiving infantformula, breast milk, a human milk fortifier, or combinations thereof.In one embodiment, the compositions are administered to preterm infantsas an enteral nutritional supplement. As used herein, the term “preterminfants” or “premature infants” means infants born after less than 37weeks gestation.

The compositions may, but need not, be nutritionally complete. Theskilled artisan will recognize “nutritionally complete” to varydepending on a number of factors including, but not limited to, age,clinical condition, and dietary intake of the subject to whom the termis being applied. In general, “nutritionally complete” means that thenutritional composition of the present invention provides adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for normal growth. As applied tonutrients, the term “essential” refers to any nutrient which cannot besynthesized by the body in amounts sufficient for normal growth and tomaintain health and which therefore must be supplied by the diet. Theterm “conditionally essential” as applied to nutrients means that thenutrient must be supplied by the diet under conditions when adequateamounts of the precursor compound is unavailable to the body forendogenous synthesis to occur.

The composition which is “nutritionally complete” for the preterm infantwill, by definition, provide qualitatively and quantitatively adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for growth of the preterm infant. Thecomposition which is “nutritionally complete” for the term infant will,by definition, provide qualitatively and quantitatively adequate amountsof all carbohydrates, lipids, essential fatty acids, proteins, essentialamino acids, conditionally essential amino acids, vitamins, minerals,and energy required for growth of the term infant. The skilled artisanwill recognize the term “term infant” as referring to infants born afterat least 37 weeks gestation, and more commonly between 37 and 42 weeksgestation. The composition which is “nutritionally complete” for a childwill, by definition, provide qualitatively and quantitatively adequateamounts of all carbohydrates, lipids, essential fatty acids, proteins,essential amino acids, conditionally essential amino acids, vitamins,minerals, and energy required for growth of a child given the specificage and developmental stage of the child.

The nutritional composition may be provided in any form known in theart, including a powder, a gel, a suspension, a paste, a solid, aliquid, a liquid concentrate, or a ready-to-use product. In onepreferred embodiment, the nutritional composition is an infant formula,especially an infant formula adapted for use as sole source nutritionfor an infant. In another embodiment, the nutritional composition is aninfant formula adapted for use as sole source nutrition for preterminfants. In other embodiments, the nutritional composition may be afollow-up formula, growing-up milk, milk modifier and combinationsthereof.

In the preferred embodiments, the nutritional product disclosed hereinmay be administered enterally. As used herein, “enteral” means throughor within the gastrointestinal, or digestive, tract, and “enteraladministration” includes oral feeding, intragastric feeding,transpyloric administration, or any other introduction into thedigestive tract.

Suitable fat or lipid sources for practicing the present invention maybe any known or used in the art, including but not limited to, animalsources, e.g., milk fat, butter, butter fat, egg yolk lipid; marinesources, such as fish oils, marine oils, single cell oils; vegetable andplant oils, such as corn oil, canola oil, sunflower oil, soybean oil,palmolein, coconut oil, high oleic sunflower oil, evening primrose oil,rapeseed oil, olive oil, flaxseed (linseed) oil, cottonseed oil, higholeic safflower oil, palm stearin, palm kernel oil, wheat germ oil;medium chain triglyceride oils and emulsions and esters of fatty acids;and any combinations thereof.

Bovine milk protein sources useful in practicing the present inventioninclude, but are not limited to, milk protein powders, milk proteinconcentrates, milk protein isolates, nonfat milk solids, nonfat milk,nonfat dry milk, whey protein, whey protein isolates, whey proteinconcentrates, sweet whey, acid whey, casein, acid casein, caseinate(e.g. sodium caseinate, sodium calcium caseinate, calcium caseinate) andany combinations thereof.

In one embodiment, the proteins are provided as intact proteins. Inother embodiments, the proteins are provided as a combination of bothintact proteins and partially hydrolyzed proteins, with a degree ofhydrolysis of between about 4% and 10%. In yet another embodiment, theprotein source may be supplemented with glutamine-containing peptides.

In a particular embodiment of the invention, the whey:casein ratio ofthe protein source is similar to that found in human breast milk. In anembodiment, the protein source comprises from about 40% to about 80%whey protein. In another embodiment, the protein source may comprisefrom about 20% to about 60% caseins.

In one embodiment of the invention, the nutritional composition maycontain one or more probiotics. The term “probiotic” means amicroorganism that exerts beneficial effects on the health of the host.Any probiotic known in the art may be acceptable in this embodimentprovided it achieves the intended result. In a particular embodiment,the probiotic may be selected from Lactobacillus species, Lactobacillusrhamnosus GG, Bifidobacterium species, Bifidobacterium longum, andBifidobacterium animalis subsp. lactis BB-12.

If included in the composition, the amount of the probiotic may varyfrom about 10⁴ to about 10¹⁰ colony forming units (cfu) per kg bodyweight per day. In another embodiment, the amount of the probiotic mayvary from about 10⁶ to about 10⁹ cfu per kg body weight per day. In yetanother embodiment, the amount of the probiotic may be at least about10⁶ cfu per kg body weight per day.

In an embodiment, the probiotic(s) may be viable or non-viable. As usedherein, the term “viable”, refers to live microorganisms. The term“non-viable” or “non-viable probiotic” means non-living probioticmicroorganisms, their cellular components and/or metabolites thereof.Such non-viable probiotics may have been heat-killed or otherwiseinactivated but retain the ability to favorably influence the health ofthe host. The probiotics useful in the present invention may benaturally-occurring, synthetic or developed through the geneticmanipulation of organisms, whether such new source is now known or laterdeveloped.

The nutritional composition contains one or more prebiotics. The term“prebiotic” as used herein refers to indigestible food ingredients whichexert health benefits upon the host. Such health benefits may include,but are not limited to, selective stimulation of the growth and/oractivity of one or a limited number of beneficial gut bacteria,stimulation of the growth and/or activity of ingested probioticmicroorganisms, selective reduction in gut pathogens, and favorableinfluence on gut short chain fatty acid profile. Such prebiotics may benaturally-occurring, synthetic, or developed through the geneticmanipulation of organisms and/or plants, whether such new source is nowknown or developed later. Prebiotics useful in the present invention mayinclude oligosaccharides, polysaccharides, and other prebiotics thatcontain fructose, xylose, soya, galactose, glucose and mannose. Morespecifically, prebiotics useful in the present invention may includelactulose, lactosucrose, raffinose, gluco-oligosaccharide, inulin,polydextrose, polydextrose powder, fructo-oligosaccharide,isomalto-oligosaccharide, soybean oligosaccharides, lactosucrose,xylo-oligosacchairde, chito-oligosaccharide, manno-oligosaccharide,aribino-oligosaccharide, siallyl-oligosaccharide, fuco-oligosaccharide,galacto-oligosaccharide, and gentio-oligosaccharides.

In an embodiment, the total amount of prebiotics present in thenutritional composition may be from about 1.0 g/L to about 10.0 g/L ofthe composition. As noted, the total amount of prebiotics present in thenutritional composition may be from about 2.0 g/L and about 8.0 g/L ofthe composition. At least 20% of the prebiotics should comprisegalacto-oligosaccharide

In addition to galacto-oligosaccharide, the prebiotic composition canalso comprise polydextrode (PDX). If polydextrose is used as aprebiotic, the amount of polydextrose in the nutritional compositionmay, in an embodiment, be within the range of from about 1.0 g/L toabout 4.0 g/L.

The amount of galacto-oligosaccharide in the nutritional compositionmay, in an embodiment, be from about 0.2 mg/100 Kcal to about 1.0 mg/100Kcal. In another embodiment, the amount of galacto-oligosaccharide inthe nutritional composition may be from about 0.1 mg/100 Kcal to about0.5 mg/100 Kcal. If polydextrose is used as a prebiotic, the amount ofpolydextrose in the nutritional composition may, in an embodiment, bewithin the range of from about 0.1 mg/100 Kcal to about 0.5 mg/100 Kcal.In another embodiment, the amount of polydextrose may be about 0.3mg/100 Kcal. In a particular embodiment, galacto-oligosaccharide andpolydextrose are supplemented into the nutritional composition in atotal amount of about 0.6 mg/100 Kcal.

The nutritional formulation of the invention contains a source of longchain polyunsaturated fatty acids (LCPUFAs) which comprisedocosahexanoic acid (DHA). Other suitable LCPUFAs include, but are notlimited to, α-linoleic acid, γ-linoleic acid, linoleic acid, linolenicacid, eicosapentanoic acid (EPA) and arachidonic acid (ARA).

In one embodiment, the nutritional composition is supplemented with bothDHA and ARA. In this embodiment, the weight ratio of ARA:DHA may be fromabout 1:3 to about 9:1. In one embodiment of the present invention, thisratio is from about 1:2 to about 4:1.

The amount of long chain polyunsaturated fatty acids in the nutritionalcomposition may vary from about 5 mg/100 kcal to about 100 mg/100 kcal,more preferably from about 10 mg/100 kcal to about 50 mg/100 kcal.

The nutritional composition may be supplemented with oils containing DHAand ARA using standard techniques known in the art. For example, DHA andARA may be added to the formula by replacing an equivalent amount of anoil, such as high oleic sunflower oil, normally present in the formula.As another example, the oils containing DHA and ARA may be added to theformula by replacing an equivalent amount of the rest of the overall fatblend normally present in the formula without DHA and ARA.

If utilized, the source of DHA and ARA may be any source known in theart such as marine oil, fish oil, single cell oil, egg yolk lipid, andbrain lipid. In some embodiments, the DHA and ARA are sourced from thesingle cell Martek oil, DHASCO®, or variations thereof. The DHA and ARAcan be in natural form, provided that the remainder of the LCPUFA sourcedoes not result in any substantial deleterious effect on the infant.Alternatively, the DHA and ARA can be used in refined form.

In an embodiment of the present invention, sources of DHA and ARA aresingle cell oils as taught in U.S. Pat. Nos. 5,374,567; 5,550,156; and5,397,591, the disclosures of which are incorporated herein in theirentirety by reference. However, the present invention is not limited toonly such oils.

In a particular embodiment, the composition of the invention is amilk-based nutritional composition which provides physiochemical andphysiological benefits. As is known in the art, bovine milk proteincomprises two major components: acid soluble whey protein and acidinsoluble casein, with the latter representing about 80% of the totalprotein content of bovine milk. Upon entering the acidic environment ofthe stomach, casein precipitates and complexes with minerals formingsemi-solid curds of varying size and firmness. Softer, smaller curds areeasier for the body to digest than larger, harder curds. Curd formationmay be an important consideration in the development of nutritionalcompositions, including, but not limited to infant formulas, medicalfoods, and premature infant formulas. In an embodiment of the presentinvention, the composition of the invention provides a nutritionalcomposition having softer and smaller curds than standard infantformulas.

As discussed above, the nutritional composition of the invention alsocontains functional proteins; in one specific embodiment, thecomposition contains TGF-β. In a particular embodiment of the invention,the level of TGF-β in the inventive composition is from about 0.0150(pg/μg) ppm to about 0.1 (pg/μg) ppm. In another embodiment, the levelof TGF-β in the inventive composition is from about 0.0225 (pg/μg) ppmto about 0.0750 (pg/μg) ppm.

In a particular embodiment of the invention, the level of TGF-β in theinventive composition is from about 2500 pg/mL to about 10,000 pg/mLcomposition, more preferably from about 3000 pg/mL to about 8000 pg/mL.

In one embodiment, the ratio of TGF-β1:TGF-β2 in the inventivecomposition is in the range of about 1:1 to about 1:20, or, moreparticularly, in the range of about 1:5 to about 1:15.

In some embodiments, the bioactivity of TGF-β in a nutritionalcomposition is enhanced by the addition of a bioactive-enhanced wheyfraction. In an embodiment, this bioactive-enhanced whey fraction may bea whey protein concentrate. In a particular embodiment, the whey proteinconcentrate may be Salibra® 800, available from Glanbia Nutritionals. Inanother embodiment, the whey protein concentrate may be Nutri Whey 800,available from DMV International. In yet another embodiment, the wheyprotein concentrate may be Salibra-850, available from GlanbiaNutritionals. In still another embodiment, the whey protein concentratemay be Prolacta Lacatalis WPI90, available from Lactilus IndustrieU.S.A., Inc. In a further embodiment, the whey protein concentrate maybe supplied by MG Nutritionals.

In some embodiments, the composition of the invention induces oraltolerance. As used herein, the term “oral tolerance” refers to thespecific suppression of cellular and/or humoral immune responses to anantigen by prior administration of the antigen by the oral route. Oraltolerance affects the responsiveness of the local immune system in theintestinal mucosa itself, thus preventing hypersensitivity reactions tofood proteins that could otherwise elicit potent inflammatory reactionsin the gut. Development of oral tolerance is an important component inappropriate mucosal immune function. Oral antigens, like food, foodproteins or commensal bacteria, are normally processed in a manner thatresults in a regulated immune response. This response does not injurethe host and results in systemic hypo-responsiveness in subsequent oralchallenge with the same food antigen. Thus oral tolerance isestablished. Oral tolerance can fail, however, in response to thedevelopment and pathogenesis of several immunologically based diseases,including inflammatory bowel disease, Crohn's disease, and ulcerativecolitis. In a particular embodiment, the combination of TGF-β and theprebiotics of the present invention may synergistically contribute tothe induction and/or promotion of oral tolerance to antigens incircumstances where oral tolerance has previously failed. In someembodiments, the induction of oral tolerance may be enhanced byadministration of the composition of the invention. In otherembodiments, the oral tolerance acquired by a subject may be promoted ormaintained by administration of the composition of the invention.

The following examples describe various embodiments of the presentdisclosure. Other embodiments within the scope of the claims herein willbe apparent to one skilled in the art from consideration of thespecification or practice of the invention as disclosed herein. It isintended that the specification, together with the examples, beconsidered to be exemplary only, with the scope and spirit of theinvention being indicated by the claims which follow the examples. Inthe examples, all percentages are given on a weight basis unlessotherwise indicated.

Example 1

This example illustrates another embodiment of a powdered infant formulaof the present disclosure.

Ingredients

Ingredient Amount per 100 kg Lactose, Grind A 34.277 kg Palm Olein Oil12.267 kg Coconut Oil 5.452 kg Soy Oil 5.452 kg High Oleic Sunflower Oil4.089 kg Non-Fat Dry Milk, Medium-Heat, Spray Dried 14.670 kg WheyProtein Concentrate, 35% Protein, 14.670 kg Super SackGalacto-Oligosaccharide Syrup (77% solids, 6.840 kg 44% fiber) CalciumGluconate, Monohydrate 1.606 kg Single Cell Arachidonic Acid Oil 0.347kg Single Cell Docosahexaenoic Acid Oil 0.238 kg Choline Bitartrate0.228 kg Potassium Chloride 0.198 kg Sodium Chloride 24.780 g MagnesiumOxide, Light 22.794 g L-Carnitine 9.911 g Ascorbic Acid 146.436 gInositol 37.278 g Corn Syrup Solids 33.159 g Taurine 31.659 g Dryvitamin E Tocopheryl Acetate, 50% 23.625 g Vitamin A Palmitate, DryBeadlets, CW 7.356 g Dispersible, 250 Niacinamide 6.051 g Vitamin K1 DryPhytonadione USP Powder, 5.097 g 1% Calcium Pantothenate 3.084 g VitaminB₁₂, 0.1% in starch 1.983 g Biotin Trituration, 1% 1.503 g Vitamin D₃Powder 0.906 g Riboflavin 0.705 g Thiamine Hydrochloride 0.561 gPyridoxine Hydrochloride 0.483 g Folic Acid 0.114 g Corn Syrup Solids192.187 g Ferrous Sulfate, Heptahydrate 49.600 g Ascorbic Acid 6.213 gMalto-Dextrin 146.096 g Cytidine 5′-Monohposphate, Free Acid 11.604 gUridine 5′-Monophosphate, Disodium Salt 3.419 g Adenosine5′-Monophosphate, Free Acid 2.711 g Guanosine 5′-Monophosphate, DisodiumSalt 2.170 g Lactose, Grind A 138.017 g Zinc Sulfate, Monohydrate 16.422g Corn Syrup Solids 3.616 g Sodium Selenite, Anhydrous 0.018 g CupricSulfate, Powder (CuSO₄•5H₂O) 1.688 g Manganese Sulfate, Monohydrate0.239 g

Proximate Analysis

Grams per 100 mL Grams per at Normal Caloric 100 g Dilution DistributionProtein 10.84 1.47 8.34 Fat 28.57 3.89 49.50 Carbohydrate 54.87 7.4642.16 Ash 2.70 0.37 Moisture 3.02 89.9 Calories 510 69.4

Nutrients

Nutrient Quantities per 100 Calories Calories 100 Protein, g 2.1 Fat, g5.6 Carbohydrates, g 10.6 Ash, g 0.6 Water, mL (normal dilution) 133Linoleic Acid, mg 900 α-Linolenic Acid, mg 85 Arachidonic Acid, mg 25Docosahexaenoic Acid, mg 17 Vitamin A, IU 300 Vitamin D, IU 60 VitaminE, IU 2 Vitamin K, mcg 8 Thiamin, mcg 80 Riboflavin, mcg 140 Vitamin B₆,mcg 60 Vitamin B₁₂, mcg 0.3 Niacin, mcg 1000 Folic Acid, mcg 16Pantothenic Acid, mcg 500 Biotin, mcg 3 Vitamin C, mg 12 Choline, mg 24Inositol, mg 6 Taurine, mg 6 Carnitine, mg 2 Calcium, mg 78 Phosphorus,mg 43 Magnesium, mg 8 Iron, mg 1.8 Zinc, mg 1 Manganese, mcg 15 Copper,mcg 75 Iodine, mcg 10 Sodium, mg 27 Potassium, mg 108 Chloride, mg 63Selenium, mcg 2.8 Galacto-oligosaccharide 0.6 AMP Equivalents, mg 0.5CMP Equivalents, mg 2.5 GMP Equivalents, mg 0.3 UMP Equivalents, mg 0.9Nucleotide Equivalents, mg 4.2

To prepare 1 liter of product at standard dilution (20 kcal/fl. oz.),136 grams of powder was mixed with 895.2 grams of water. To prepare 1quart of product at standard dilution, 128.7 grams of powder was mixedwith 847.2 grams water.

Upon reconstitution, the infant formula described in this examplecontains approximately 4 g/L of galacto-oligosaccharide and has an ARAlevel of 25 mg/100 kcal. The formula contains 5.6 g fat/100 kcal, toachieve a fat content which is similar to human milk. The formulaadditionally has a low buffer strength.

All pH adjustments with regard to this infant formula were made withsolutions of potassium hydroxide. The specific gravity of the formula is1.03117.

Example 2

This example illustrates an embodiment of a powdered infant formula ofthe present disclosure.

Ingredients

Ingredient Amount per 100 kg Lactose, Grind A 35.119 kg Palm Olein Oil12.264 kg Coconut Oil 5.451 kg Soy Oil 5.451 kg High Oleic Sunflower Oil4.088 kg Non-Fat Dry Milk, Medium-Heat, Spray Dried 14.667 kg WheyProtein Concentrate, 35% Protein, 14.667 kg Super SackGalacto-Oligosaccharide Syrup (77% solids, 3.477 kg 44% fiber)Polydextrose Power (96% total solids, 96% 1.770 kg carbohydrate, 86%fiber) Calcium Gluconate, Monohydrate 1.606 kg Single Cell ArachidonicAcid Oil 0.347 kg Single Cell Docosahexaenoic Acid Oil 0.238 kg CholineBitartrate 0.228 kg Potassium Chloride 0.198 kg Sodium Chloride 24.780 gMagnesium Oxide, Light 22.790 g L-Carnitine 9.910 g Ascorbic Acid156.687 g Inositol 39.887 g Corn Syrup Solids 35.478 g Taurine 33.875 gDry vitamin E Tocopheryl Acetate, 50% 25.279 g Vitamin A Palmitate, DryBeadlets, CW 7.871 g Dispersible, 250 Niacinamide 6.475 g Vitamin K1 DryPhytonadione USP Powder, 5.454 g 1% Calcium Pantothenate 3.299 g VitaminB₁₂, 0.1% in starch 2.122 g Biotin Trituration, 1% 1.608 g Vitamin D₃Powder 0.969 g Riboflavin 0.755 g Thiamine Hydrochloride 0.601 gPyridoxine Hydrochloride 0.518 g Folic Acid 0.122 g Corn Syrup Solids192.187 g Ferrous Sulfate, Heptahydrate 49.600 g Ascorbic Acid 6.213 gMalto-Dextrin 146.096 g Cytidine 5′-Monohposphate, Free Acid 11.604 gUridine 5′-Monophosphate, Disodium Salt 3.419 g Adenosine5′-Monophosphate, Free Acid 2.711 g Guanosine 5′-Monophosphate, DisodiumSalt 2.170 g Lactose, Grind A 138.017 g Zinc Sulfate, Monohydrate 16.422g Corn Syrup Solids 3.616 g Sodium Selenite, Anhydrous 0.018 g CupricSulfate, Powder (CuSO₄•5H₂O) 1.688 g Manganese Sulfate, Monohydrate0.239 g

Proximate Analysis

Grams per 100 mL Grams per at Normal Caloric 100 g Dilution DistributionProtein 10.84 1.47 8.50 Fat 28.57 3.89 50.67 Carbohydrate 54.87 7.4640.83 Ash 2.70 0.37 Moisture 3.02 89.9 Calories 508 69.1

Nutrients

Nutrient Quantities per 100 Calories Calories 100 Protein, g 2.1 Fat, g5.6 Carbohydrates, g 10.6 Ash, g 0.6 Water, mL (normal dilution) 133Linoleic Acid, mg 900 α-Linolenic Acid, mg 85 Arachidonic Acid, mg 25Docosahexaenoic Acid, mg 17 Vitamin A, IU 300 Vitamin D, IU 60 VitaminE, IU 2 Vitamin K, mcg 8 Thiamin, mcg 80 Riboflavin, mcg 140 Vitamin B₆,mcg 60 Vitamin B₁₂, mcg 0.3 Niacin, mcg 1000 Folic Acid, mcg 16Pantothenic Acid, mcg 500 Biotin, mcg 3 Vitamin C, mg 12 Choline, mg 24Inositol, mg 6 Taurine, mg 6 Carnitine, mg 2 Calcium, mg 78 Phosphorus,mg 43 Magnesium, mg 8 Iron, mg 1.8 Zinc, mg 1 Manganese, mcg 15 Copper,mcg 75 Iodine, mcg 10 Sodium, mg 27 Potassium, mg 108 Chloride, mg 63Selenium, mcg 2.8 Polydextrose 0.3 Galacto-oligosaccharide 0.3 AMPEquivalents, mg 0.5 CMP Equivalents, mg 2.5 GMP Equivalents, mg 0.3 UMPEquivalents, mg 0.9 Nucleotide Equivalents, mg 4.2

To prepare 1 liter of product at standard dilution (20 kcal/fl. oz.),136 grams of powder was mixed with 895.2 grams of water. To prepare 1quart of product at standard dilution, 128.7 grams of powder was mixedwith 847.2 grams water.

Upon reconstitution, the infant formula described in this examplecontains approximately 2 g/L of galacto-oligosaccharide and 2 g/L ofpolydextrose. The infant formula has an ARA level of 25 mg/100 kcal. Theformula contains 5.6 g fat/100 kcal, to achieve a fat content which issimilar to human milk. The formula additionally has a low bufferstrength.

All pH adjustments with regard to this infant formula were made withsolutions of potassium hydroxide. The specific gravity of the formula is1.03117.

All references cited in this specification, including withoutlimitation, all papers, publications, patents, patent applications,presentations, texts, reports, manuscripts, brochures, books, internetpostings, journal articles, periodicals, and the like, are herebyincorporated by reference into this specification in their entireties.The discussion of the references herein is intended merely to summarizethe assertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

Although preferred embodiments of the invention have been describedusing specific terms, devices, and methods, such description is forillustrative purposes only. The words used are words of descriptionrather than of limitation. It is to be understood that changes andvariations may be made by those of ordinary skill in the art withoutdeparting from the spirit or the scope of the present invention, whichis set forth in the following claims. In addition, it should beunderstood that aspects of the various embodiments may be interchangedboth in whole or in part. For example, while methods for the productionof a commercially sterile liquid nutritional supplement made accordingto those methods have been exemplified, other uses are contemplated.Therefore, the spirit and scope of the appended claims should not belimited to the description of the preferred versions contained therein.

1. A nutritional composition comprising a. a lipid or fat; b. a proteinsource; c. about 5 to about 100 mg/100 kcal of a source of long chainpolyunsaturated fatty acids which comprises docosahexanoic acid; d.about 0.1 to about 1 mg/100 kcal of a prebiotic composition, wherein theprebiotic composition comprises at least 20% of an oligosaccharide whichcomprises galacto-oligosaccharide; e. about 0.015 to about 0.1 (pg/μg)ppm of TGF-β.
 2. The nutritional composition of claim 1, wherein thesource of long chain polyunsaturated fatty acids further comprisesarachidonic acid.
 3. The nutritional composition of claim 2, wherein theratio of arachidonic acid to docosahexanoic acid is from about 1:3 toabout 9:1.
 4. The nutritional composition of claim 1, wherein the lipidor fat is present at a level of up to about 7 g/100 kcal.
 5. Thenutritional composition of claim 4, wherein the lipid or fat is presentat a level of about 3 g/100 kcal to about 7 g/100 kcal.
 6. Thenutritional composition of claim 1, wherein the protein source ispresent at a level of up to about 5 g/100 kcal.
 7. The nutritionalcomposition of claim 6, wherein the protein source is present at a levelof about 1 to about 5 g/100 kcal.
 8. The nutritional composition ofclaim 1, wherein the prebiotic composition further comprisespolydextrose.
 9. The nutritional composition of claim 1, which furthercomprises at least one probiotic.
 10. The nutritional composition ofclaim 9, wherein the probiotic is selected from the group consisting ofBifidobacteria spp., Lactobacillus spp and combinations thereof.
 11. Thenutritional composition of claim 1, wherein the level of TGF-β is fromabout 0.0225 (pg/μg) ppm to about 0.0750 (pg/μg) ppm.
 12. Thenutritional composition of claim 1, wherein the level of TGF-β is fromabout 2500 pg/mL to about 10,000 pg/mL composition.
 13. The nutritionalcomposition of claim 1, wherein the ratio of TGF-β1:TGF-β2 is about 1:1to about 1:20.
 14. The nutritional composition of claim 1, which furthercomprises a bioactive-enhanced whey fraction.
 15. The nutritionalcomposition of claim 1, wherein the combination of TGF-β and theprebiotic synergistically contributes to the induction of oral toleranceto antigens.
 16. The nutritional composition of claim 1, which comprisesan infant composition.
 17. A method for promoting oral tolerance to foodprotein antigens in a subject, comprising administering to the subject acomposition comprising a. a lipid or fat; b. a protein source; c. about5 to about 100 mg/100 kcal of a source of long chain polyunsaturatedfatty acids which comprises docosahexanoic acid; d. about 0.1 to about 1mg/100 kcal of a prebiotic composition, wherein the prebioticcomposition comprises at least 20% of an oligosaccharide which comprisesgalacto-oligosaccharide; e. about 0.015 to about 0.1 (pg/μg) ppm ofTGF-β.
 18. The method of claim 17, wherein the prebiotic compositionfurther comprises polydextrose.
 19. The method of claim 17, wherein thecomposition further comprises at least one probiotic.
 20. The method ofclaim 19, wherein the probiotic is selected from the group consisting ofBifidobacteria spp., Lactobacillus spp and combinations thereof.
 21. Themethod of claim 17, wherein the level of TGF-β is from about 0.0225(pg/μg) ppm to about 0.0750 (pg/μg) ppm.
 22. The method of claim 17,wherein the level of TGF-β is from about 2500 pg/mL to about 10,000pg/mL composition.
 23. The method of claim 17, wherein the ratio ofTGF-β1:TGF-β2 is about 1:1 to about 1:20.
 24. The method of claim 17,wherein the nutritional composition further comprises abioactive-enhanced whey fraction.
 25. The method of claim 17, whereinthe subject is an infant.